The present invention relates generally to the glycoprotein lactoferrin, its production, purification, and uses.
Lactoferrin plays an important role in iron transport and utilization in humans. A glycoprotein containing about 6% sugar and having a total molecular weight of about 80,000 daltons, human lactoferrin is capable of binding two ferric ions with high affinity. Although lactoferrin binds iron tightly, the binding is reversible so that the metal is available upon demand to cells with a need for this essential element. The metal is captured by the side chains of specific amino acids: two tyrosines, one histidine and one aspartate which in combination form a cleft in the surface of the protein. That portion of the protein which contains the aforesaid four amino acids and forms the cleft is termed the xe2x80x9ciron-binding domain.xe2x80x9d Each natural lactoferrin molecule has two iron-binding domains.
Human milk is high in lactoferrin content. The high degree of iron absorption from human milk is manifested in a low incidence of iron deficiency anemia among breast fed infants compared to infants fed with cow""s milk. Accordingly, lactoferrin is a key protein for healthy development of infants. Lactoferrin also plays an important role in cell-mediated host immunity. It is present in high concentrations in all bodily secretions, such as tears, sweat, and ciliated respiratory mucous. Because it sequesters iron, lactoferrin can neutralize pathogenic microorganisms by preventing them from obtaining necessary iron at the site of entry, thereby preventing the spread of infection.
Although iron is an essential material in humans, excess iron in the body can induce pathological conditions as well. Chronic iron overload, known as hemosiderosis, is characterized by a greater than normal local or generalized deposition of iron within certain body tissues. Lactoferrin helps to manage the balance of free iron in the body to prevent occurrence of such pathological states in healthy individuals.
The severely limited amount of human milk, the major source of human lactoferrin, restricts lactoferrin production. Furthermore, production of lactoferrin from human milk presents a risk factor of infectious contamination. That is, it could carry with it a potentially lethal contaminant, such as the human immunodeficiency virus (HIV) or another undesirable agent.
Accordingly, the present invention provides for the cloning and expression of human lactoferrin using recombinant DNA techniques. The lactoferrin of the present invention is free of naturally occurring contaminants, e.g., proteins and viruses, that would be detrimental to the recipient.
In one embodiment of the present invention there is provided a gene comprising a DNA molecule encoding human lactoferrin protein. More particularly, the DNA molecule comprises the nucleotide sequence (Seq. ID No. 1) and the protein comprises the amino acid sequence (Seq. ID No. 2) as substantially depicted in FIG. 3. An expression system is provided for expressing the gene encoding the protein. Preferably, the expression system is a plasmid. Also described herein, is a host cell line transformed with the gene of the present invention, i.e., the gene encoding human lactoferrin. Preferably the host cell organism is a eukaryotic cell.
In another embodiment of the invention, there is provided a method of producing human lactoferrin comprising the steps of (a) isolating a gene encoding human lactoferrin; (b) transforming a host cell with the gene; (c) culturing the transformed cells which express the gene product; and (d) collecting lactoferrin from the cells.
In a further embodiment of the invention there is described a chromatography method for purifying lactoferrin protein, and other proteins, comprising the steps of (a) contacting a substance with a first adsorbent to obtain adsorbed and non-absorbed fractions; (b) eluting the adsorbed fraction with an eluant; and (c) contacting the adsorbed fraction with a second adsorbent, wherein the improvement comprises equilibrating the second adsorbent with the eluant followed by contacting the eluate containing the adsorbed fraction with the second adsorbent.
In still a further embodiment of the invention there is provided a method for inhibiting microbial growth in a mammal comprising topically or systemically applying to a subject a therapeutically effective amount of lactoferrin having less than about 25% metal loading; a method for inhibiting iron deficiency in a mammal comprising orally administering a therapeutically effective amount of lactoferrin having at least about 35% iron loading; a nutritional supplement comprising an iron-loaded human lactoferrin having at least about 35% metal loading and a nutritionally acceptable carrier or adjuvant; a topical antiseptic comprising a therapeutically effective amount of lactoferrin having less than about 25% metal loading and a pharmaceutically acceptable carrier or diluent; and a method for inhibiting food spoilage comprising adding to the food an effective amount of lactoferrin having less than about 25% metal loading.